Use of periostin as a novel biomarker

ABSTRACT

The invention provides, in certain embodiments, a method of detecting an indicator of renal injury or renal disease. The method entails assaying a urine sample for periostin, wherein the presence of periostin at an elevated level indicates the presence and/or degree of renal injury or renal disease. Also provided, are methods of determining progression of these conditions, as well as methods of determining subjects&#39; response to treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/571,211, filed Aug. 9, 2012, which is a divisional of U.S.application Ser. No. 12/924,608, filed Sep. 29, 2010, which claims thebenefit of U.S. provisional application No. 61/251,248, filed Oct. 13,2009, all of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to the use of periostin as a marker for kidneyinjury and/or kidney disease.

BACKGROUND OF THE INVENTION

Chronic kidney disease (CKD) is increasing worldwide and is emerging asa major global health threat.[1] Accurately assessing and monitoringrenal function is of critical importance in patients with CKD.Currently, kidney injury is clinically tested by serum creatinine, serumcystatin-C, or urine protein or albumin. These are markers that measurekidney functional loss, but not kidney cellular injury. A few urineproteins are also measured as markers of tubulointerstitial injury, butthey are either not generally used clinically (e.g.,N-acetyl-beta-D-glucosaminidase) or are experimental tests (NGAL, KIM1,Interleukin-18 (IL-18)).

Periostin was initially identified in osteoblasts and acts as anadhesion molecule during bone formation, supports osteoblastic cell lineattachment, and is involved in cell survival, proliferation, migration,and differentiation.[3-6] It is induced in processes and pathologiesincluding cardiac embryogenesis, adult cardiac disease, metastaticdisease, and tumor suppression. [7] Evidence in these tissues suggeststhat periostin may play a fundamental role in tissue remodeling [8,9],and in disease of the cardiovascular system.[10-12] Periostin is inducedin the kidney during nephrogenesis, but it is not observed in adultkidney under normal conditions.[13] It also may accelerate cyst growthand promote interstitial remodeling in polycystic kidney disease(PKD).[14]

SUMMARY OF THE INVENTION

In certain embodiments, the invention provides a method of detecting anindicator of renal injury or renal disease. The method entails assayinga urine sample for periostin, wherein the presence of periostin at anelevated level indicates the presence and/or degree of renal injury orrenal disease. In various embodiments, periostin is detected as adiagnostic indicator of renal injury or renal disease; an indicator ofprogression, remission, or relapse of renal injury or renal disease; oran indicator of response to treatment for renal injury or renal disease.In specific embodiments, periostin is detected as an indicator ofepithelial mesenchymal transition (EMT).

In illustrative embodiments, the urine sample comprises a human urinesample. The urine sample can be, for example, centrifuged urine orurinary exosomes. The human can be, e.g., a human patient known to have,or suspected of having, renal injury or renal disease. The renal diseasecan be acute or chronic.

In particular embodiments, the invention provides a method of detectingan indicator of a subject's response to treatment for renal injury orrenal disease. The method entails assaying a urine sample obtained froma subject after initiation of treatment for renal injury or renaldisease for periostin, wherein the level of periostin is positivelycorrelated with the degree of renal injury or renal disease. In certainembodiments, a baseline level of periostin is measured prior toinitiation of treatment for renal injury or renal disease. In avariation of such embodiments, the periostin level of the urine sampleafter initiation of treatment is compared to the baseline level ofperiostin. A decrease in the periostin level of the urine sample afterinitiation of treatment, as compared to the baseline level of periostin,indicates that the subject is responding to the treatment. The methodcan also entail, in some embodiments, performing one or more additionalassays of periostin.

Any of the methods described herein can additionally entail detectingone or more additional indicators of renal injury or disease selectedfrom the group consisting of serum creatinine, serum cystatin-C, urineprotein, urine albumin, urine N-acetyl-beta-D-glucosaminidase, urineNGAL, IL-18, urine KIM1, and hematopoietic growth factor inducibleneurokinin-1 (HGFIN).

In any of these methods, the periostin can be detected by any suitablemethod, such as, e.g., an immunoassay, HPLC, and mass spectroscopy. Theperiostin can, for example, be detected in an assay wherein theperiostin becomes labeled with a detectable label. In some embodiments,periostin is detected in an assay wherein the periostin is transformedfrom a free state to a bound state by forming a complex with anotherassay component. In illustrative embodiments, periostin is detected inan assay wherein periostin initially present in a soluble phase becomesimmobilized on a solid phase. The assay can, in some embodiments, entailfractionating the sample to separate periostin from at least one othersample component. In illustrative embodiments, periostin is detected inan assay wherein periostin becomes embedded in a separation medium. Infurther illustrative embodiments, periostin is detected in an assaywherein periostin is volatilized.

Any of the methods described herein can additionally entail recordingthe periostin level, and/or a diagnosis based at least in part on theperiostin level, in a patient medical record. This recordation caninclude recording the periostin level in a computer-readable medium. Invarious embodiments, the patient medical record is maintained by alaboratory, physician's office, a hospital, a health maintenanceorganization, an insurance company, or a personal medical recordwebsite. In certain embodiments, a diagnosis, based at least in part onthe periostin level, is recorded on or in a medic alert article, such asa card, worn article, or radiofrequency identification (RFID) tag.

Any of the methods described herein can additionally entail informingthe subject of a result of the periostin assay and/or of a diagnosisbased at least in part on the periostin level. These methods can alsoentail prescribing, initiating, and/or altering prophylaxis and/ortherapy. In particular embodiments, the methods entail ordering and/orperforming one or more additional assays. For example, if the periostinlevel determined in an initial periostin assay is not elevated, and theadditional assay comprises an additional periostin assay (e.g., at alater date). If the periostin level determined in an initial periostinassay is elevated, an additional periostin assay can be carried out (forconfirmation of the elevated level) or a different assay can be carriedout, e.g., to detect a different biomarker of renal injury or disease.

In any of the methods described here, periostin is detected as part of adifferential diagnosis of renal injury or disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-B and 1C1-C6. Renal periostin increases after 5/6Nx in rats.(A) Periostin mRNA expression increased over time after 5/6Nx in RKcompared to control kidney tissue in samples in which the infarct tissuewas excised. The expression of 18 S was used as an internal control. (B)Immunoblotting analysis for periostin was also increased in RK comparedto control kidneys. (C) Periostin immunostaining was not detected incortical control rat kidney (C1). In contrast, representative sectionsof kidney tissues at 2 days, 2 weeks, and 4 weeks displayed cytoplasmicstaining for periostin, most prominently in the apical portion oftubular cells, with stronger and more diffuse tubular cell staining at 2and 4 weeks. There also was periostin staining in casts and/or insloughed cells in the tubular lumina (C3, C4, arrows). There was noglomerular staining for periostin (C3) (C1-4 Original magnification:400×). (C5) Renal tubules demonstrated apical periostin in the 2 weekRK. Tubules contained luminal sloughed cells and cellular debris whichstained strongly for periostin (arrows) (Original magnification: 600×).(C6) 4 week RK had periostin positive interstitial cells (arrows) whichfrequently were in the periadventitial area around arteries andarterioles. (Original magnification: 400×). * P<0.05 vs. control group,#P<0.05 vs. 2 days after 5/6Nx group.

FIGS. 2A-B and 2C1-C6. Renal periostin expression increased afterdiabetes induction and UUO in mice. (A) Renal periostin mRNA expressionincreased after 2 months of SZ injection in DBA2J mice compared tocontrol kidneys. The expression of 18 S was used as an internalcontrol.* P<0.05 vs. control. (B) Renal periostin protein was increasedin SZ-DM DBA2J mice compared to control DBA2J mice at 8 week and 16weeks. * P<0.05 vs. DBA2J mice control kidneys 8 weeks, #P<0.05 vs.DBA2J mice control kidneys 16 weeks. (C) Representative micrographsshowed positive periostin immunostaining in renal tubules of SZ-DM at 2months and UUO at 5 days and 14 days. (C1) Control, 8 wks; (C2) Control,16 weeks; (C3) SZ-DM, 16 weeks; (C4) Control; (C5) UUO, Day 5; (C6) UUO,Day 14. (Original magnification: 200×).

FIG. 3A-F. Periostin localizes exclusively to tubular cells of thedistal nephron after 5/6Nx. Paraffin-embedded sections were doublelabeled with antibodies against periostin (red, A, D) and either distalnephron marker PNA lectin (green, B) or proximal nephron marker PHA-Electin (green, E). Periostin co-localized with PNA staining exclusivelyin the distal nephron (C), but never with PHA-E staining in the proximalnephron (F). Merged images show periostin in red and PNA or PHA-E ingreen (Original magnification: 200×). Cell nuclei were stained with DAPI(C and F).

FIGS. 4A1-A4 and 4B1-B12. Periostin induces EMT phenotype. (A)E-cadherin expression is lost in distal nephron tubules expressingcytoplasmic periostin after 5/6Nx. The serial sections show virtuallymutually exclusive immunofluorescence staining patterns for cytoplasmicperiostin (red, A1) and E-cadherin (green, A2) in RK tissues 4 weeksafter 5/6Nx. The section was counterstained with DAPI to visualize thecell nuclei and the tubules (merge, A3). Sequential sections also showthat tubules expressing either periostin or E-cadherin both continued toexpress PNA lectin (A4), demonstrating that both are being expressed indistal nephron tubules. (B) Periostin, FSP1, and MMP9 are co-expressedin RK 2 days, 2 weeks, and 4 weeks after 5/6Nx. Serial sections ofremnant kidney were stained for Periostin (B1, B4, B7, B10), FSP1 (B2,B5, B8, B11), and MMP9 (B3, B6, B9, B12) at all time points after 5/6Nx;2 days (B1-3); 2 weeks (B4-6); and 4 weeks (B7-12) B1-9: Staining ofMMP9 and FSP1 showed co-localization with periostin in renal tubularepithelium, and in luminal sloughed tubular cells and cytoplasmicfragments at all times after 5/6Nx. Cell and luminal cellular debrisstain for all three proteins at 2 weeks (arrows) (Originalmagnification: 600×). B10-12: Interstitial cells in the 4 week remnantkidney also stain for periostin, FSP1 and MMP9 (arrows) (Originalmagnification 400×).

FIGS. 5A and 5B1-B4. Periostin-producing cells increase expression ofEMT markers. Cell lysates from parental cells, transfected empty vectorcells (control), transfected periostin vector cells and co-transfectedperiostin with SureSilencing siRNA vector cells were employed to examineMMP9, FSP1, and E-cadherin expression (A). Results are shown in (B) as aratio of β-actin level; (B1) Periostin; (B2) MMP9; (B3) FSP1; (B4)E-cadherin. MDCT cells expressing periostin dramatically increased MMP9and FSP1 expression, a hallmark for mesenchymal cell. E-cadherinexpression was also decreased by the periostin transgene in the cells.Co-transfected periostin and SureSilencing siRNA vector cells expressedreduced levels of periostin protein. Reduced periostin expressionresulted in a restoration of E-cadherin and partial reduction of MMP9and FSP1 expression. * P<0.05 vs. parental, control and periostin+siRNAgroup.

FIG. 6A-6B. Urine periostin excretion rate increase after 5/6Nx in theRK model of progressive renal injury and in patients with proteinuricrenal diseases and non-proteinuric renal disease. (A) Western blottinganalysis for urine periostin was performed on individual rats prior to5/6 Nx and after 2 days, 2 weeks, and 4 weeks (n=3 at each time point).Each lane was loaded with 2% of the total urinary flow rate for each ratsample. Urine creatinine was measured and used to control forconcentration. Representative Western blots are shown. Experiments wereperformed in triplicate. * P<0.05 vs. pre surgery group, #P<0.05 vs. 2days after 5/6Nx group. (B) In lightly centrifuged urine treated andstored with protease inhibitors, then thawed for the assay, 90 kDa urineperiostin was detectable in patients with various proteinuric glomerulardiseases, but not in controls (0.03 ml urine). With urine collectedidentically, in patients with non-proteinuric PKD but not in controls,90 kDa urine periostin is also clearly detectable. C, control; DN,Diabetic nephropathy; LN, Lupus nephritis; FSGS, Focal and segmentalglomerulosclerosis; PKD, Polycystic kidney disease.

FIGS. 7A, 7B1-B4 and 7C. Urine periostin ELISA has high performance indiagnosing CKD and it correlates with decline of GFR and increment ofurine NGAL. (A) Urine periostin/creatinine measured by ELISA is higherin patients with progressive proteinuric renal disease (n=21) and in PKD(n=5) than in healthy controls (n=20). Individual values for eachpatient and control represents the average of at least triplicatetesting. The median values for patients with progressive proteinuricdisease (2473.58 pg/mg), and PKD (9504.94 pg/mg) were not significantlydifferent from each other, but were significantly higher than forhealthy controls (0 pg/mg). (B) Univariate baseline statisticalcorrelations (Sperman coefficient) of urinary periostin. Significantcorrelations were evidenced with estimated GFR (B1), serum creatinine(B2), and urinary NGAL (B4). (C) Receiver operating characteristicscurves of urinary periostin and NGAL considering CKD as status variable.The area under the curve for urinary periostin and NGAL was 0.96 (95%CI, 0.91 to 1.02) and 0.86 (95% CI, 0.75 to 0.97), respectively. Bothurinary periostin and NGAL areas were statistically different withrespect to that of diagnostic reference line (P<0.001). On the contrary,the difference between the two biomarker areas was non-significant(P=0.09).

FIG. 8A-8D. Urine periostin is measurable before a rise in serumcreatinine is discernible in renal tissue from a patient with LN inwhich tubular atrophy is present. (A) Renal biopsy showing proliferativelupus glomerulonephritis with an area of established tubular atrophy(arrow) (Jones stain, Original magnification: ×200). (B) Immunoblottingdemonstrating 90 kDa urine periostin in lightly centrifuged urine, butnone in control. (C) Periostin immunostaining (brown; H&E counterstain,Original magnification: ×200) shows cytoplasmic tubular cell expressionincluding expression in sloughed luminal cell fragment (arrow). (D)Tubular cells with heavy diffuse cytoplasmic periostin immunostaining(arrow) (Original magnification: ×400).

DETAILED DESCRIPTION Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

“Biological samples” that can be assayed using the methods of thepresent invention include biological fluids, such as kidney tissue andcells, whole blood, blood leukocytes, serum, and urine.

As used herein with reference to periostin, the term “elevated level”refers to a level in a biological sample that is higher than a normallevel or range. The normal level or range for periostin is defined inaccordance with standard practice. Thus, the level measured in aparticular biological sample will be compared with the level or range oflevels determined in similar normal samples. In this context, “normaltissue” is tissue from an individual with no detectable renal diseaseand/or renal injury. The level of periostin is said to be “elevated”where the periostin is normally undetectable (i.e, the normal level inthe tissue is zero), but is detected in a test sample, as well as wherethe periostin is present in the test sample at a higher than normallevel or range.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain(VL)” and “variable heavy chain (VH)” refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)2 dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (sFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked VH-VL heterodimer which may beexpressed from a nucleic acid including VH- and VL-encoding sequenceseither joined directly or joined by a peptide-encoding linker. Huston,et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH andVL are connected to each as a single polypeptide chain, the VH and VLdomains associate non-covalently. The scFv antibodies and a number ofother structures converting the naturally aggregated, but chemicallyseparated light and heavy polypeptide chains from an antibody V regioninto a molecule that folds into a three dimensional structuresubstantially similar to the structure of an antigen-binding site areknown to those of skill in the art (see e.g., U.S. Pat. Nos. 5,091,513,5,132,405, and 4,956,778).

The term “specific binding” is defined herein as the preferentialbinding of binding partners to another (e.g., two polypeptides, apolypeptide and nucleic acid molecule, or two nucleic acid molecules) atspecific sites. The term “specifically binds” indicates that the bindingpreference (e.g., affinity) for the target molecule/sequence is at least2-fold, more preferably at least 5-fold, and most preferably at least10- or 20-fold over a non-specific target molecule (e.g. a randomlygenerated molecule lacking the specifically recognized site(s)).

As used herein, the phrase “periostin becomes labeled with a detectablelabel” refers to the binding of a label or labeled moiety to periostin,directly or indirectly, via one or more additional moieties.

As used with reference to periostin, a “free state” refers to the stateof periostin before contact with any assay component. This termencompasses periostin bound to one or more sample components. The term“bound state” is used to describe periostin bound to one or more assaycomponent(s) to form a complex.

The term “medical record” or “patient medical record” refers to anaccount of a patient's examination and/or treatment that typicallyincludes one or more of the following: the patient's medical history andcomplaints, the physician's physical findings, the results of diagnostictests and procedures, and patient medications and therapeuticprocedures. A medical record is typically made by one or more physiciansand/or physicians' assistants and is a written, transcribed or otherwiserecorded record and/or history of various illnesses or injuriesrequiring medical care, and/or inoculations, and/or allergies, and/ortreatments, and/or prognosis, and/or frequently health information aboutparents, siblings, and/or occupation. The record may be reviewed by aphysician in diagnosing the condition.

As used herein, the term “worn article” refers to any article that canbe worn on a subject's body, including, but not limited to, a tag,bracelet, necklace, arm band, or head band.

As used herein, the term “differential diagnosis” refers to thedetermination of which of two or more diseases with similar symptoms islikely responsible for a subject's symptom(s), based on an analysis ofthe clinical data.

In General

Early recognition is important to slowing kidney disease progression,maintaining quality of life, and improving outcomes. Accuratelyassessing and monitoring renal function is of critical importance inpatients with kidney disease. Biomarkers for early kidney disease andfor kidney disease progression are currently not sensitive or specific.More sensitive and specific biomarkers are needed to diagnose kidneyinjury at an early stage and to assess response (either injurious ofstate or remission) to treatments.

In certain embodiments, the invention provides methods of detectingperiostin as a novel biomarker of renal injury and/or renal disease.These methods entail assaying a biological sample for periostin, whereinthe level of periostin is positively correlated with renal injury and/orrenal disease. In various embodiments, these methods are useful indiagnosing acute kidney injury as well as ongoing kidney injury,obviating the need for an initial or one or more serial kidney biopsiesin some clinical situations. In other embodiments, these methods can beemployed to assess response to therapy and/or identify relapse of kidneyinjury. Furthermore, these methods are unique in that they have thecapability of non-invasively assessing, in a quantitative manner, aparticular pathophysiologic process (e.g., epithelial mesnchymaltransformation (EMT)) that is a major driver of progressive renalinjury. Measurement of periostin, e.g., in urine, provides a surrogatemeasure of EMT and therefore a valuable tool for assessing the successof treatments for kidney diseases.

Sample Collection and Processing

The assay methods of the invention are generally carried out onbiological samples derived from an animal, preferably a mammal, and morepreferably a human.

The methods of the invention can be carried out using any sample thatmay contain soluble periostin, periostin in exosomes, or periostinmoieties, including its intracellular, transmembrane, or extracellularmoieties or any peptide fraction thereof Convenient samples include, forexample, blood, blood cells, serum, plasma, kidney cells, urinaryexosomes, and urine.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution or concentrated, if desired. Any of a number of standardaqueous buffer solutions and/or protease inhibitors, employing any of avariety of buffers, such as phosphate, Tris, or the like, atphysiological pH, can be used.

Assaying for Periostin

Periostin can be detected and quantified by any of a number of methodswell known to those of skill in the art for polypeptide detection. Thesemay include analytic biochemical methods such as electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,mass spectroscopy and the like, or various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunohistochemistry, affinity chromatography, immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, and the like.

In one embodiment, periostin is detected/quantified in anelectrophoretic polypeptide separation (e.g. a 1- or 2-dimensionalelectrophoresis). Means of detecting polypeptides using electrophoretictechniques are well known to those of skill in the art (see generally,R. Scopes (1982) Polypeptide Purification, Springer-Verlag, N.Y.;Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to PolypeptidePurification, Academic Press, Inc., N.Y.).

A variation of this embodiment utilizes a Western blot (immunoblot)analysis to detect and quantify the presence of periostin in the sample.This technique generally comprises separating sample polypeptides by gelelectrophoresis on the basis of molecular weight, transferring theseparated polypeptides to a suitable solid support (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with antibodies that specifically bind theanalyte. Antibodies that specifically bind to the analyte may bedirectly labeled or alternatively may be detected subsequently usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to a domain of the primary antibody.

In certain of the above-described embodiments, the sample and/orperiostin is transformed in some manner in the course of the assay. Forexample, the sample may be fractionated such that periostin is separatedfrom at least one other sample component. The periostin can be recoveredin a liquid fraction or can be detected while embedded in a separationmedium, such as a gel. For mass spectroscopy, periostin is volatilizedfor detection.

In a preferred embodiment, periostin is detected and/or quantified inthe biological sample using any of a number of well-known immunoassays(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). For a general review of immunoassays, see also Methods inCell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. AcademicPress, Inc. New York (1993); Basic and Clinical Immunology 7th Edition,Stites & Terr, eds. (1991).

Conventional immunoassays often utilize a “capture agent” tospecifically bind to and often immobilize the analyte on a solid phase.In preferred embodiments, the capture agent is an antibody.

Immunoassays also typically utilize a labeled detection agent tospecifically bind to and label the binding complex formed by the captureagent and the analyte. The labeled detection agent may itself be one ofthe moieties making up the antibody/analyte complex. Alternatively, thelabeled detection agent may be a third moiety, such as another antibody,that specifically binds to the capture agent/analyte complex. Otherpolypeptides capable of specifically binding immunoglobulin constantregions, such as polypeptide A or polypeptide G may also make up thelabeled detection agent. These polypeptides are normal constituents ofthe cell walls of streptococcal bacteria. They exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, generally Kronval, et al. (1973) J. Immunol.,111: 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542).

Preferred immunoassays for detecting the target polypeptide(s) areeither competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of captured analyte is directly measured. Incompetitive assays, the amount of analyte in the sample is measuredindirectly by measuring the amount of an added (exogenous) labeledanalyte displaced (or competed away) from a capture agent by the analytepresent in the sample. In one competitive assay, a known amount of, inthis case, labeled periostin is added to the sample, and the sample isthen contacted with a capture agent. The amount of labeled periostinbound to the antibody is inversely proportional to the concentration ofperiostin present in the sample.

In illustrative embodiments, periostin is measured in urine using a“dipstick” assay.

The assays of this invention are scored (as positive or negative orquantity of analyte) according to standard methods well known to thoseof skill in the art. The particular method of scoring will depend on theassay format and choice of label. For example, a Western Blot assay canbe scored by visualizing the colored product produced by the enzymaticlabel. A clearly visible colored band or spot at the correct molecularweight is scored as a positive result, while the absence of a clearlyvisible spot or band is scored as a negative. The intensity of the bandor spot can provide a quantitative measure of analyte concentration.

Antibodies

Antibodies useful in the immunoassay methods of the invention includepolyclonal and monoclonal antibodies. Polyclonal antibodies are raisedby injecting (e.g., subcutaneous or intramuscular injection) animmunogen into a suitable non-human mammal (e.g., a mouse or a rabbit).Generally, the immunogen should induce production of high titers ofantibody with relatively high affinity for the target antigen.

If desired, the antigen may be conjugated to a carrier protein byconjugation techniques that are well known in the art. Commonly usedcarriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The conjugate is then used toimmunize the animal.

The antibodies are then obtained from blood samples taken from theanimal. The techniques used to produce polyclonal antibodies areextensively described in the literature (see, e.g., Methods ofEnzymology, “Production of Antisera With Small Doses of Immunogen:Multiple Intradermal Injections,” Langone, et al. eds. (Acad. Press,1981)). Polyclonal antibodies produced by the animals can be furtherpurified, for example, by binding to and elution from a matrix to whichthe target antigen is bound. Those of skill in the art will know ofvarious techniques common in the immunology arts for purification and/orconcentration of polyclonal, as well as monoclonal, antibodies see, forexample, Coligan, et al. (1991) Unit 9, Current Protocols in Immunology,Wiley Interscience.

For many applications, monoclonal antibodies (mAbs) are preferred. Thegeneral method used for production of hybridomas secreting mAbs is wellknown (Kohler and Milstein (1975) Nature, 256:495). Briefly, asdescribed by Kohler and Milstein, the technique entailed isolatinglymphocytes from regional draining lymph nodes of five separate cancerpatients with either melanoma, teratocarcinoma or cancer of the cervix,glioma or lung, (where samples were obtained from surgical specimens),pooling the cells, and fusing the cells with SHFP-1. Hybridomas werescreened for production of antibody that bound to cancer cell lines.Confirmation of specificity among mAbs can be accomplished using routinescreening techniques (such as the enzyme-linked immunosorbent assay, or“ELISA”) to determine the elementary reaction pattern of the mAb ofinterest.

As used herein, the term “antibody” encompasses antigen-binding antibodyfragments, e.g., single chain antibodies (scFv or others), which can beproduced/selected using phage display technology. The ability to expressantibody fragments on the surface of viruses that infect bacteria(bacteriophage or phage) makes it possible to isolate a single bindingantibody fragment, e.g., from a library of greater than 10¹⁰ nonbindingclones. To express antibody fragments on the surface of phage (phagedisplay), an antibody fragment gene is inserted into the gene encoding aphage surface protein (e.g., pIII) and the antibody fragment-pIII fusionprotein is displayed on the phage surface (McCafferty et al. (1990)Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137).

Since the antibody fragments on the surface of the phage are functional,phage-bearing antigen-binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990) Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20-fold-1,000,000-fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000-fold in one roundcan become 1,000,000-fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552-554). Thus, even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after as few as three to four rounds ofselection. Thus only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment, natural VHand VL repertoires present in human peripheral blood lymphocytes areisolated from unimmunized donors by PCR. The V-gene repertoires can bespliced together at random using PCR to create a scFv gene repertoirewhich can be cloned into a phage vector to create a library of 30million phage antibodies (Id.). From a single “naïve” phage antibodylibrary, binding antibody fragments have been isolated against more than17 different antigens, including haptens, polysaccharides, and proteins(Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor, and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). The antibody fragments are highly specific for theantigen used for selection and have affinities in the 1 nM to 100 nMrange (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Griffiths et al.(1993) EMBO J. 12: 725-734). Larger phage antibody libraries result inthe isolation of more antibodies of higher binding affinity to a greaterproportion of antigens.

As those of skill in the art readily appreciate, antibodies can beprepared by any of a number of commercial services (e.g., Berkeleyantibody laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).

Solid Phase

For embodiments of the invention that employ a solid phase as a supportfor the capture agent, the solid phase can be any suitable porousmaterial with sufficient porosity to allow access by reagents and asuitable surface affinity to bind a capture agent. Microporousstructures are generally preferred, but materials with gel structure inthe hydrated state may be used as well. Useful solid supports include:natural polymeric carbohydrates and their synthetically modified,crosslinked, or substituted derivatives, such as agar, agarose,cross-linked alginic acid, substituted and cross-linked guar gums,cellulose esters, especially with nitric acid and carboxylic acids,mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins; natural hydrocarbon polymers, such aslatex and rubber; synthetic polymers which may be prepared with suitablyporous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer. All of these materials maybe used in suitable shapes, such as films, sheets, or plates, or theymay be coated onto, bonded, or laminated to appropriate inert carriers,such as paper, glass, plastic films, fabrics, or the like.

The porous structure of nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents including monoclonalantibodies. Nylon also possesses similar characteristics and also issuitable.

Porous solid phases useful in the invention can be in the form of sheetsof thickness from about 0.01 to 0.5 mm, e.g., about 0.1 mm. The poresize may vary within wide limits, and is preferably from about 0.025 toabout 15 microns, especially from about 0.15 to about 15 microns.

Preferred solid phase materials for flow-through assay devices includefilter paper such as a porous fiberglass material or other fiber matrixmaterials. The thickness of such material is not critical and will be amatter of choice, largely based upon the properties of the sample oranalyte being assayed, such as the fluidity of the biological sample.

Alternatively, the solid phase can constitute microparticles.Microparticles useful in the invention can be selected by one skilled inthe art from any suitable type of particulate material and include thosecomposed of polystyrene, polymethylacrylate, polypropylene, latex,polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similarmaterials.

Microparticles can be suspended in the mixture of soluble reagents andbiological sample or can be retained and immobilized by a supportmaterial. In the latter case, the microparticles on or in the supportmaterial are not capable of substantial movement to positions elsewherewithin the support material.

The methods of the present invention can be adapted for use in systemsthat utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S. App.No. 425,651 and U.S. Pat. No. 5,089,424, which correspond to publishedEPO App. Nos. EP 0 425 633 and EP 0 424 634, respectively, and U.S. Pat.No. 5,006,309.

In particular embodiments, the solid phase includes one or moreelectrodes. Capture agent(s) can be affixed, directly or indirectly, tothe electrode(s). In one embodiment, for example, capture agents can beaffixed to magnetic or paramagnetic microparticles, which are thenpositioned in the vicinity of the electrode surface using a magnet.Systems in which one or more electrodes serve as the solid phase areuseful where detection is based on electrochemical interactions.Exemplary systems of this type are described, for example, in U.S. Pat.No. 6,887,714 (issued May 3, 2005). The basic method is describedfurther below with respect to electrochemical detection.

The capture agent can be attached to the solid phase by adsorption onthe porous material, where it is retained by hydrophobic forces.Alternatively, the surface of the solid phase can be activated bychemical processes that cause covalent linkage of the capture agent tothe support.

To change or enhance the intrinsic charge of the solid phase, a chargedsubstance can be coated directly onto the solid phase material or ontomicroparticles which then are retained by a solid phase material. Ioncapture procedures for immobilizing an immobilizable reaction complexwith a negatively charged polymer, described in U.S. App. No. 150,278,corresponding to EP Publication No. 0326100, and U.S. App. No. 375,029(EP Publication No. 0406473), can be employed according to the presentinvention to affect a fast solution-phase immunochemical reaction. Inthese procedures, an immobilizable immune complex is separated from therest of the reaction mixture by ionic interactions between thenegatively charged polyanion/immune complex and the previously treated,positively charged porous matrix and detected by using any of a numberof signal-generating systems, including, e.g., chemiluminescent systems,as described in U.S. App. No. 921,979, corresponding to EPO PublicationNo. 0 273,115.

If the solid phase is silicon or glass, the surface must generally beactivated prior to attaching the specific binding partner. Activatedsilane compounds such as triethoxy amino propyl silane (available fromSigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (AldrichChemical Co., Milwaukee, Wis.), and (3-mercapto-propyl)-trimethoxysilane (Sigma Chemical Co., St. Louis, Mo.) can be used to introducereactive groups such as amino-, vinyl, and thiol, respectively. Suchactivated surfaces can be used to link the capture directly (in thecases of amino or thiol), or the activated surface can be furtherreacted with linkers such as glutaraldehyde, bis(succinimidyl)suberate,SPPD 9 succinimidyl 3-[2-pyridyldithio]propionate), SMCC(succinimidyl-4-[Nmaleimidomethyl]cyclohexane-1-carboxylate), SIAB(succinimidyl [4iodoacetyl]aminobenzoate), and SMPB (succinimidyl4-[1maleimidophenyl]butyrate) to separate the capture agent from thesurface. Vinyl groups can be oxidized to provide a means for covalentattachment. Vinyl groups can also be used as an anchor for thepolymerization of various polymers such as poly-acrylic acid, which canprovide multiple attachment points for specific capture agents. Aminogroups can be reacted with oxidized dextrans of various molecularweights to provide hydrophilic linkers of different size and capacity.Examples of oxidizable dextrans include Dextran T-40 (molecular weight40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons),Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M(molecular weight 2,000,000 daltons) (all of which are available fromPharmacia, Piscataway, N.J.), or Ficoll (molecular weight 70,000daltons; available from Sigma Chemical Co., St. Louis, Mo.).Additionally, polyelectrolyte interactions can be used to immobilize aspecific capture agent on a solid phase using techniques and chemistriesdescribed U.S. App. No. 150,278, filed Jan. 29, 1988, and U.S. App. No.375,029, filed Jul. 7, 1989, each of which is incorporated herein byreference.

Other considerations affecting the choice of solid phase include theability to minimize non-specific binding of labeled entities andcompatability with the labeling system employed. For, example, solidphases used with fluorescent labels should have sufficiently lowbackground fluorescence to allow signal detection.

Following attachment of a specific capture agent, the surface of thesolid support may be further treated with materials such as serum,proteins, or other blocking agents to minimize non-specific binding.

Labeling Systems

As discussed above, many immunoassays according to the invention employa labeled detection agent.

Detectable labels suitable for use in the detection agents of thepresent invention include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, orchemical means. Useful labels in the present invention include magneticbeads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, texasred, rhodamine, green fluorescent protein, and the like, see, e.g.,Molecular Probes, Eugene, Oreg., USA), chemiluminescent compounds suchas acridinium (e.g., acridinium-9-carboxamide), phenanthridinium,dioxetanes, luminol and the like, radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C,or ³²P), catalysts such as enzymes (e.g., horse radish peroxidase,alkaline phosphatase, beta-galactosidase and others commonly used in anELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The label can be attached to the detection agent prior to, or during, orafter contact with the biological sample. So-called “direct labels” aredetectable labels that are directly attached to or incorporated intodetection agents prior to use in the assay. Direct labels can beattached to or incorporated into detection agents by any of a number ofmeans well known to those of skill in the art.

In contrast, so-called “indirect labels” typically bind to the detectionagent at some point during the assay. Often, the indirect label binds toa moiety that is attached to or incorporated into the detection agentprior to use. Thus, for example, an antibody used as a detection agent(a “detection antibody”) can be biotinylated before use in an assay.During the assay, an avidin-conjugated fluorophore can bind thebiotin-bearing detection agent, to provide a label that is easilydetected.

In another example of indirect labeling, polypeptides capable ofspecifically binding immunoglobulin constant regions, such aspolypeptide A or polypeptide G, can also be used as labels for detectionantibodies. Such polypeptides can thus be labeled and added to the assaymixture, where they will bind to the detection antibody.

Some labels useful in the invention may require the use of an indicatorreagent to produce a detectable signal. In an ELISA, for example, anenzyme label (e.g., beta-galactosidase) will require the addition of asubstrate (e.g., X-gal) to produce a detectable signal.

Periostin Levels

Once determined, a periostin level can be recorded in a patient medicalrecord. In certain embodiments, the methods of the invention includemaking a diagnosis, often a differential diagnosis, based at least inpart on the periostin level. This diagnosis can also be recorded in apatient medical record. For example, in various embodiments, thediagnosis of renal injury and/or renal disease (acute or chronic) isrecorded in a medical record. The medical record can be in paper formand/or can be maintained in a computer-readable medium. The medicalrecord can be maintained by a laboratory, physician's office, ahospital, a health maintenance organization, an insurance company,and/or a personal medical record website. In certain embodiments, adiagnosis, based at least in part on the periostin level, is recorded onor in a medic alert article such as a card, a worn article, and/or aradiofrequency identification (RFID) tag.

In particular embodiments, the methods of the invention includeinforming the subject of a result of the periostin assay and/or of adiagnosis based at least in part on the periostin level. The patient canbe informed verbally, in writing, and/or electronically.

The methods of the invention can include prescribing, initiating, and/oraltering prophylaxis and/or therapy, e.g., for renal injury and/or renaldisease (acute or chronic). In certain embodiments, the methods canentail ordering and/or performing one or more additional assays. Forexample, if the periostin level is determined to be within a normalrange (i.e., not elevated), the periostin assay may be repeated to ruleout a false negative result, and/or one or more additional periostinassays may be performed to monitor the subject's status. If theperiostin level is determined to be elevated, it may be desirable repeatthe periostin assay to rule out a false positive result. In certainembodiments, it will be desirable to assay another indicator of, e.g.,renal injury and/or renal disease (acute or chronic), to confirm adiagnosis. Exemplary indicators of renal injury or disease include serumcreatinine, serum cystatin-C, urine protein, urine albumin, urineN-acetyl-beta-D-glucosaminidase, urine NGAL, IL-18, urine KIM1, andhematopoietic growth factor inducible neurokinin-1 (HGFIN). The use ofHGFIN as an indicator of renal injury or disease is described in U.S.patent application Ser. No. 12/613,385, filed Nov. 5, 2009, which ishereby incorporated by reference in its entirety and specifically forthis description. Urine periostin may be sequentially measured inpatients in whom the assay shows kidney injury in order to demonstrateremission, and in those with remission, in order to demonstrate relapseof kidney injury. In the setting of experimental drug testing, urineperiostin may be used alone or as a member of a biomarker panel todemonstrate early kidney injury either in preclinical and/or clinicaltesting.

Test Kits

The invention also provides a test kit for assaying for periostin. Testkits according to the invention include one or more reagents useful forpracticing one or more immunoassays according to the invention. A testkit generally includes a package with one or more containers holding thereagents, as one or more separate compositions or, optionally, asadmixture where the compatibility of the reagents will allow. The testkit can also include other material(s) that may be desirable from a userstandpoint, such as a buffer(s), a diluent(s), a standard(s), and/or anyother material useful in sample processing, washing, or conducting anyother step of the assay.

Test kits according to the invention preferably include instructions forcarrying out one or more of the immunoassays of the invention.Instructions included in kits of the invention can be affixed topackaging material or can be included as a package insert. While theinstructions are typically written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to, electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD ROM), and the like. As used herein, the term “instructions” caninclude the address of an internet site that provides the instructions.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Periostin: Novel Tissue and Urinary Biomarker of ProgressiveRenal Injury Induces a Coordinated Mesenchymal Phenotype in TubularCells Abstract

Background: Periostin acts as an adhesion molecule during boneformation. Knowledge of expression of periostin in kidney injury isstill scanty.

Methods: We investigated periostin function and expression in vitro ofdistal nephron tubular cells (DT), in Sprague-Dawley rats after 5/6nephrectomy (Nx), in DBA2J mice after streptozotocin-induced diabetes(SZ-DM), and in the urine of chronic kidney disease (CKD) patients.

Results: Periostin was identified by microarray and confirmed byreal-time PCR in renal tissue after 5/6Nx, and SZ-DM demonstratinggeneralizability of the periostin increment in renal injury. Periostinwas expressed predominantly in DT and in tubule cells shed into thelumen. In affected DT after 5/6Nx, periostin expression appeared denovo, the epithelial cell adhesion molecule E-cadherin becameundetectable, and tubule cells displayed the mesenchymal marker proteinsfibroblast specific protein-1 (FSP1) and matrix metalloproteinase-9(MMP9). To assess whether periostin plays a direct role in renal tubularepithelial mesenchymal transition (EMT), we overexpressed periostin incultured DT. Overexpression dramatically increased MMP9 and FSP1protein, and decreased E-cadherin protein expression. In addition, theeffect of periostin on the renal tubular EMT was also blocked byperiostin siRNA transfection. Urine periostin excretion increased overtime after 5/6Nx, and it was also excreted in the urine of CKD patients.Urine periostin ELISA at a cutoff value of 32.66 pg/mg creatininedemonstrated sensitivity and specificity for distinguishing patientswith progressive CKD from healthy people (92.3%, and 95.0%,respectively).

Conclusions: These data demonstrate that periostin is a mediator andmarker of EMT, and a promising tissue and urine biomarker for kidneyinjury in experimental models and in clinical renal disease.

Introduction

The aim of the present study was to investigate periostin expression andfunction in animal models of kidney disease and in CKD patients.

Subjects and Methods

Animals

Sprague Dawley rats (N=18) underwent 5/6nephrectomy (Nx) (N=9) byunilateral Nx and ligation of 2/3 of the vessels to the contralateralkidney or sham Nx. Rats were sacrificed at 2 days, 2 weeks, and 4 weeksafter surgery. Diabetes was induced in DBA2J mice by intraperitonealinjection of streptozotocin 40 mg/kg/day for 5 days as previouslydescribed with minor modifications.[15] At 2 months, renal tissues wereharvested. DBA2J mice were subjected to unilateral ureteral obstruction(UUO) of left kidney and renal tissues were harvested at 5 and 14 days.All procedures were performed in accordance with the guidelinesestablished by the National Research Council Guide for the Care and Useof Laboratory Animals.

Gene Array Analysis

AFFYMETRIC GENE CHIP 230_(—)2 expression analysis was used to comparethe transcription profiles between normal kidneys and remnant kidney(RK) at 2 days, 2 weeks and 4 weeks after 5/6Nx. Total RNA from 3 RK ateach time point and 3 normal kidneys were labeled and hybridized toAFFYMETRIC GENE CHIPs. Data were expressed as the average differencesbetween the perfect match and mismatch probes for the periostin gene.

Collection of Human Urine

CKD subjects were recruited from our outpatient Nephrology clinic.Random urine samples were collected from proteinuric CKD patients (n=21)and non proteinuric CKD patients with PKD (n=5) and stored at −80° C.with protease inhibitors until assayed. Control samples were collectedfrom healthy volunteers (n=20) who have normal renal function.

Quantitative Real Time-Polymerase Chain Reaction (RT-PCR) Analysis

Total RNA was isolated from rat control kidneys and RK at 2 days, 2weeks and 4 weeks after 5/6Nx and DBA2J mice control kidneys andstreptozotocin-induced diabetes (SZ-DM) at 2 months. RT-PCR withrelative quantification of periostin copy number in relation to 18sribosomal RNA transcripts was carried out using the following primers:periostin forward TGGTGTTGTCCATGTCATCGA (SEQ ID NO:1); and periostinreverse TGTGAAGTGACCGTCTCTTCCA (SEQ ID NO:2). All PCRs were run in anABI 7900 Sequence Detection System (Applied Biosystems).

Immunohistochemistry

Four micron sections of formalin-fixed, paraffin-embedded tissue weredeparaffinized and rehydrated. Endogenous peroxidase activity wasquenched by incubating the slides in endogenous enzyme block solution,and subsequently at 4° C. for overnight with the primary polyclonalperiostin antibody, fibroblast specific protein-1 (FSP1) antibody andmatrix metalloproteinase-9 (MMP9) antibody. Next, the sections wereincubated with dextran polymer conjugated with horseradish peroxidaseand affinity isolated immunoglobulin for 30 minutes at room temperature.

Immunofluorescence

Deparaffinized rat kidney sections prepared as described were doublelabeled with a primary rabbit polyclonal periostin antibody and eitherfluorescence-conjugated peanut agglutinin (PNA) lectin antibody specificfor distal nephron tubules (DT), fluorescence-conjugated phaseolusvulgaris erythroagglutinin (PHA-E) lectin antibody specific for proximalnephron tubules, and/or FITC-conjugated monoclonal E-cadherin antibody.In addition, using serial sections and PNA as a marker of DT, wecompared the localization of periostin and E-cadherin in the DT.Indirect primary antibody was followed with goat anti-rabbit IgGconjugated to Texas Red.

Immunoblotting Analysis

Frozen kidney tissue and cell lysates were standardized by proteinconcentration, and a total of 30-100 μg of protein per well was loaded.Spot urine was collected from rats, patients, and healthy volunteers.Two percent of the urinary volume for each rat sample and 0.03 ml urinefor each human sample was subjected to immunoblotting analysis. Theprocedure was done with a standard protocol as described previously.[16]

Urine Periostin Analysis by ELISA

96-well microplates were coated overnight with 1 μg/ml (0.1 μg per well)of anti-periostin antibody. Plates were washed three times with 0.05%Tween 20 in PBS then blocked with Reagent Diluent for at least one hour.100 μl of all standards and patient samples was added to the 96-wellplate and incubated for 2 hours. After a 1 hours incubation with arabbit polyclonal antibodies to periostin, 20 minutes incubation withdextran polymer conjugated with horseradish peroxidase, and 20 minutesincubation with substrate solution, stop solution was added to eachwell. Periostin absorbances were calculated by making measurements at450 nm and correcting for plate artifact at 570 nm. Periostinconcentrations were calculated based on a log-transformed standardcurve.

Urine Neutrophil Gelatinase-Associated Lipocalin (NGAL) Analysis byELISA

The urine NGAL ELISA was performed using a commercially available assay(NGAL Rapid ELISA Kit 037; Bioporto, Grusbakken, Denmark) thatspecifically detects urine NGAL. The assay was performed as per themanufacturer's protocol.

Generation of Periostin-Producing Mouse Distal Convoluted Tubule (MDCT)Cells and RNA Interference

Full-length mouse periostin cDNA was subcloned into a pCMV-SPORT6(Thermo Scientific, Huntsville, Ala.). All the plasmids were purifiedwith the Qiagen Midiprep kit. One day before transfection experiment,6×10⁵ immortalized MDCT cells, kindly provided by Dr. Peter Friedman,were plated on each well of 60 mm culture dish overnight. Confluentcells (80-90%) were then transfected with the periostin construct orvector control. For knockdown of periostin expression by using RNAinterference technique, cells were co-transfected with mouse periostinplasmid and SureSilencing siRNA plasmids for mouse periostin by usingFuGENE HD transfection reagent, according to the manufacturer'sinstruction. After transfection for 24 hours, cells were lysed andprotein levels were determined by immunobloting.

Statistical Analysis

Statistical analysis was performed using SPSS, version 15. Either atwo-sample t test or Mann-Whitney rank sum test was used for continuousvariables. For multiple comparisons, ANOVA was used followed by theleast significance difference test. Spearman correlation coefficientswere used as appropriate to test correlations between urine periostinand other variables. Receiver operating characteristics (ROC) analysiswas used to calculate the area under the curve (AUC) for periostin andNGAL and to find the best cut-off values for identifying the CKD. A P≦05was considered statistically significant.

Results

Overexpressed Periostin Gene Following Renal Injury in the RK Model

Microarray Gene Set Enrichment Analysis (GSEA, Cambridge, Mass.) showedthat gene expression of periostin was significantly up-regulated in theRK inclusive of the necrotic areas: 21.91-fold at day 2, 13.32-fold atweek 2, and 14.46-fold at week 4 when compared with control kidneys. Toconfirm the microarray observation, and to determine if it is expressedexclusively in the infarct region, we additionally examined theexpression of periostin mRNA in separate RK tissues in which theinfarcted region was excised. As shown in FIG. 1A, RT-PCR revealed thatthere was a significant difference in mRNA expression of periostin inthe RK: 3.84-fold at day 2 (P=0.025), 9.57-fold at week 2 (P=0.015), and11.05-fold at week 4 (P=0.046) compared with control kidneys. Thus, theexamination of periostin mRNA in viable RK tissue without infarctedtissue unmasked a progressive increase seen in injured renal parenchymaafter 5/6Nx.

Renal Periostin Expression Increased Over Time in the RK Model

Immunoblotting and immunohistochemical analyses were performed on RKtissue after 5/6Nx compared to control kidneys to determine periostinprotein expression. FIG. 1B shows increase in renal periostin/β-actinratio each time point after 5/6Nx compared to controls (P<0.05). Asshown in FIG. 1C staining of kidney sections of RK at all timesdemonstrated periostin expression predominantly in tubular cellcytoplasm, particularly in the apical aspects, but there was noperiostin present in control cortical kidney. Detached tubular cells andcytoplasmic cell fragments sloughed into tubular lumina frequently werepositive for periostin. The intensity of the tubular cell stainingincreased between 2 days and 2 weeks after 5/6Nx and remained at 4weeks. RK had also periostin positive interstitial cells whichfrequently were in the periadventitial area around arterioles. Thus,these data confirmed that the mRNA changes observed after 5/6Nx weretranslated into increased protein expression in tubules in thenon-infarcted RK.

Overexpression of Renal Periostin in SZ-DM, and UUO

Periostin was measured by RT-PCR in renal tissue from DBA2J mice 2months after SZ or diluent injections. FIG. 2A shows a 2.66-foldincrease in periostin mRNA in the renal tissue of SZ-DM mice compared tocontrols (P=0.008). Significantly increased periostin expression wasalso detected by immunoblotting analysis in SZ-DM renal tissue comparedwith controls (FIG. 2B). As shown in FIG. 2C staining of kidney sectionsof SZ-DM and UUO demonstrated that prominent periostin was identifieddiffusely in tubular cell cytoplasm. Therefore, these data demonstratedthat renal periostin also increased in a kidney injury model lackinginfarction.

Periostin is Expressed in DT

As shown in FIG. 3, periostin was expressed in the cytoplasm of tubularepithelial cells that also stained positively for PNA lectin, indicatingperiostin expression in DT. There was no periostin identified in nephronsegments stained with the proximal tubular lectin marker PHA-E. Thusperiostin localized to the DT in the RK.

Disappearance of the Tight Junction Protein E-Cadherin in DT ExpressingPeriostin

Using serial sections, immunofluorescence analysis of the RKdemonstrated that DT retained their affinity for PNA lectin whether thetubules did or did not express periostin. However, in these PNAlectin-positive DT, the expression of E-cadherin and periostin werevirtually mutually exclusive (FIG. 4A). These studies demonstrated anassociation between the appearance of periostin in DT in the RKconcomitant with the disappearance of the DT protein E-cadherin, thelatter a marker of the tubular differentiated state and a transmembraneprotein responsible for cell-cell adhesion.

Periostin Associates with the Appearance of Renal Tubular EpithelialMesenchymal Transition (EMT) Markers

To study EMT, tissues were stained for FSP1, a cytoplasmic marker ofepithelium undergoing mesenchymal transition, and MMP9, a proteininvolved in the turnover of extracellular matrix in renal tissueremodeling. These immunohistochemical studies revealed co-staining ofMMP9 and FSP1 with periostin in affected DT cells, including sloughedcells and cytoplasmic fragments in tubular lumina, at all time pointsafter 5/6Nx (FIG. 4B). There was staining of interstitial cells forperiostin, FSP1 and MMP9 at 2 weeks with more extensive interstitialstaining at 4 weeks. These studies demonstrate an association betweenperiostin expression and the appearance of specific proteins in renaltubule indicating EMT.

In Vitro Periostin Induces Renal Tubular Mesenchymal Phenotype

We used a transfection system to introduce the periostin cDNA into MDCTcells. MDCT cells ectopically expressing periostin dramaticallyincreased MMP9 and FSP1 expression, a hallmark for mesenchymal cells.The level of MMP9 and FSP1 in parental MDCT cells and vector controlcells was barely detectable. In contrast, expression of E-cadherin tightjunction was strikingly decreased in periostin-producing cells (FIG. 5).Gene knockdown with siRNA was next applied to analyze the function ofperiostin on renal tubular EMT. MDCT cells were co-transfected with theperiostin cDNA and siRNA, and the periostin protein level was obviouslyreduced. The effect of periostin on the renal tubular MMP9 and FSP1generation and E-cadherin reduction was blocked by periostin siRNAtransfection (FIG. 5). In aggregate, the data demonstrate that periostinexpressed by MDCT cells drives the cells to undergo EMT.

Urinary Periostin Excretion Progressively Increased Over Time in the RKModel

FIG. 6A shows the time course for the urine periostin after 5/6Nx in alongitudinal experiment in which urine was collected from the sameanimals serially until their sacrifice at 4 weeks. Urine periostin wasundetectable during the control period prior to 5/6Nx. There weresignificant incremental increases in urine periostin excretion over timeafter 5/6Nx. These data show that urine periostin distinguished healthyfrom injured kidney in a categorical fashion, and excretion increasedover time with progressive chronicity of injury.

Human Urine Periostin is Detectable by Immunoblotting in CKD Patients

In FIGS. 6B-C, urine periostin is clearly detectable both in theproteinuric and non-proteinuric CKD patients. The appearance of urineperiostin in CKD patients but not in healthy controls underscores itsvalue as a potential biomarker for kidney injury in proteinuric andnon-proteinuric conditions.

Using a Quantitative ELISA, Urine Periostin is Higher in Proteinuric andNon-Proteinuric CKD Patients than in Healthy Controls

A standard curve was generated using known concentrations of recombinantperiostin resulting in a linearized R² of 0.981 (data not shown). Table1 describes the clinical characteristics of the patients.

TABLE 1 Clinical characteristics of the patients with proteinuric andnon proteinuric chronic kidney disease Serum Serum eGFR Etiology MeanAge Albumin BUN Creatinine (mL/min/ of CKD (yrs) Gender (g/dL) (mg/dL)(mg/dL) UPCR 1.73 m²) Proteinuric 46.1 ± 14.2 F = 7, 3.1 ± 0.8 49.3 ±26.3 3.1 ± 1.7 4.6 ± 2.8 35.4 ± 34.1 patients (n = 21) M = 14 DN (n =13) 52.5 ± 10.3 F = 2, 3.4 ± 0.5 60.8 ± 19.9 3.7 ± 1.5 4.0 ± 1.9 20.4 ±6.9 M = 11 GN (n = 8) 35.8 ± 14.7 F = 5, 2.6 ± 1.1 30.5 ± 25.4 2.2 ± 1.75.7 ± 3.8 59.9 ± 46.1 M = 3 LN (n = 2) 20.5 ± 2.1  F, M 2.5 ± 0.0 46.0 ±46.7 2.9 ± 3.1 4.2 ± 1.4 59.9 ± 62.9 MN (n = 3) 41.0 ± 14.4 F = 2, M 2.5± 1.2 31.6 ± 18.0 2.7 ± 1.4 7.0 ± 1.6 34.8 ± 31.2 IgMN (n = 2) 35.5 ±17.7 F = 2 2.6 ± 2.1 7.5 ± 4.9 0.7 ± 0.2 7.4 ± 7.8 113.4 ± 20.8  FSGS (n= 1) 51.0 M 3.3 42.0 2.5 1.6 28.6 Non proteinuric patients (n = 5) PKD(n = 5) 42.2 ± 12.8 F = 3, 3.6 ± 0.3 39.0 ± 22.7 3.6 ± 2.4 0.4 ± 0.228.7 ± 24.8 M = 2 BUN, Blood urea nitrogen; UPCR; Urine proteincreatinine ratio, eGFR; estimated glomerular filtration rate, DN,Diabetic nephropathy; LN, Lupus nephritis; MN, membranous nephropathy,IgMN, IgM nephropathy, FSGS, Focal and segmental glomerulosclerosis;PKD, Polycystic kidney disease

Urine periostin was measured by ELISA in proteinuric CKD (n=21), nonproteinuric CKD (n=5), healthy controls (n=20), and in an additional twopatients with non-progressive CKD (minimal change nephropathy (MCD) andWegener's granulomatosis). The median urine periostin in healthycontrols (0 pg/mg) was significantly less than in patients withproteinuric CKD (2473.58 pg/mg, p<0.001), and non-proteinuric CKD(9504.94 pg/mg, p=0.003) (FIG. 7A). There was no significant differencebetween the median values in the patients with proteinuric CKD andnon-proteinuric CKD (p=0.72). One patient had frequently relapsing MCD,but still had 1.2 gm proteinuria/24 hours at the time the urine specimenwas taken. A second patient had a history of Wegener's granulomatosis inclinical remission for over 10 years, but had 0.8 gm proteinuria/24hours and stable serum creatinine of 2 mg/dl at the time of the urineperiostin measurement. In both cases, the periostin measurements werezero.

To assess the relationship between urine periostin and renal severity,the Spearman correlation analysis was performed as appropriate. Theresults are illustrated in FIG. 7B. The urine periostin levels directlycorrelated to serum creatinine (R=0.41, P=0.03), and urine NGAL (R=0.64,P<0.001), whereas inverse significant correlations were evidenced withestimated glomerular filtration rate (GFR) (R=−0.39, P=0.04), but it didnot significantly correlate with degree of proteinuria (R=0.30,P=0.129). These data are consistent with the hypothesis that the urineperiostin measurement reflects tubular injury, and that proteinuria andurine periostin excretion are independent processes.

Urinary Periostin is High Performance in Diagnosing CKD

The ROC analysis of urine periostin and NGAL in diagnosing CKD isillustrated in FIG. 7C. AUC for urine periostin and NGAL were 0.96 (95%CI, 0.91 to 1.02) and 0.86 (95% CI, 0.75 to 0.97), respectively. Urineperiostin and NGAL areas were statistically different with respect tothat of diagnostic reference line (P<0.001), but the both biomarkerareas were non-significant different (P=0.09). For urine periostin thebest cut-off level was 32.66 pg/mg (sensitivity 92.3%, specificity95.0%), whereas for urine NGAL it was 13.73 ng/mg (sensitivity 80.8%,specificity 80.0%). Thus, urine periostin ELISA demonstrate highsensitivity and specificity for diagnosing CKD and is comparable tourinary UGAL.

Case Vignette Demonstrating the Use of Urine Periostin Measurements inClinical Practice

As a case in point, urine periostin was compared to serum creatinine indetecting kidney injury in a 20-year old woman who presented with 1month of rapid onset malar rash, myalgias, tactile fevers, and edema.Proteinuria was 3.3 gm/day. Serum creatinine was 1.0 mg/dl (range0.9-1.2 mg/dl) during a 1-week period. Serology confirmed systemic lupuserythematosus. Renal biopsy showed proliferative glomerulonephritis withareas of established tubular atrophy (FIG. 8A). Immunoblotting detectedurine periostin in lightly centrifuged urine (FIG. 8B). Periostinimmunostaining showed cytoplasmic tubular cells expression includingexpression in sloughed luminal cell fragments (FIG. 8C) and tubularcells with heavy diffuse cytoplasmic periostin immunostaining (FIG. 8D).In this clinical setting, urine periostin measurements better reflectedthe underlying tubular injury seen histopathologically better than theserum creatinine measurements.

Discussion

The present study describes the renal expression and urine excretion ofperiostin in experimental models of renal disease, and in the urine froma group of CKD patients. Urine periostin ELISA demonstrated highsensitivity and specificity for diagnosing CKD. DT expressing periostinexpressed other traditional mesenchymal proteins such as FSP1 and MMP9,but not E-cadherin. Overexpressed periostin in cultured MDCT cellsdramatically induces expression of EMT markers and reduces tightjunction E-cadherin. Moreover, after periostin siRNA transfection, renaltubular EMT was disappeared. Taken together, these data demonstrate thatperiostin is a likely marker of EMT and a promising tissue and urinebiomarker for kidney injury.

Periostin, originally identified in osteoblasts, functions as a celladhesion molecule for preosteoblasts, and participates in osteoblastrecruitment and spreading.[3-6] Periostin may contribute to renal tissueremodeling in a manner analogous to its functions in other injuredtissues.[17,18] In previously published study, periostin was localizedwithin PKD cyst epithelial cells, and was secreted into both the tubularlumina and the interstitium.[14] In our study, staining of kidneysections of all RK at all times demonstrated periostin expression innumerous DT, predominantly in the renal tubular epithelial cellcytoplasm, and in cells shed into the lumen. The intensity of the renalparenchymal staining was increased over time after 5/6Nx. Thus, the datasuggest that the de novo expression of periostin during injury and itsexcretion in urine may be common events during progressive renalfunctional decline.

A major area of research in patients with CKD is the elucidation of EMTduring renal fibrosis. Multiple reports have demonstrated elevatedperiostin levels in malignant cells that had undergone EMT andmetastasized.[19-21] In addition, one study showed that overexpressionof periostin in a tumorigenic epithelial cell line inducedfibroblast-like transformation with increased expression of vimentin,epidermal growth factor receptor, MMP9, and evidence for increased cellmigration, and adhesion, indicative of EMT.[22] In agreement with thesepreviously reported studies conducted on neoplastic tissues, this studyalso demonstrates that overexpressed periostin in cultured MDCT cellsdramatically induced the appearance of the mesenchymal markers MMP9 andFSP1, and the decrease of the epithelial cell marker E-cadherin. Thecombination of increased MMP9 turning over basement membrane anddecreased E-cadherin diminishing cell-cell adhesion, likely contributesto DT cell sloughing, and indicates that renal tubular cell periostinexpression is a marker of EMT. Previous studies have demonstrated thattubular cells expressing proteins that contribute to extracellularmatrix turnover during EMT may migrate to the tubulointerstitium.[23]While renal epithelium cells can acquire mesenchymal markers in vitro,they do not directly contribute to interstitial myofibroblast cells invivo.[24] Thus, the study reported herein suggests that tubular cellsexpressing a mesenchymal phenotype also are at risk of losing cell-celland cell-matrix attachments and sloughing into the tubular lumen.

In conclusion, these studies demonstrate that periostin in the urine isa measure of the loss of renal tubular cells that have adopted amesenchymal phenotype in response to diverse renal injuries acrossspecies. Its histopathologic expression patterns in the kidney in situsuggest that periostin may participate in the pathogenesis of CKD as asignaling molecule.

REFERENCES

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What is claimed is:
 1. A method of detecting an indicator of renalinjury or renal disease, the method comprising assaying a urine samplefor periostin, wherein the presence of periostin at an elevated levelindicates the presence and/or degree of renal injury or renal disease.2. The method of claim 1, wherein periostin is detected as an indicatorselected from the group consisting of a diagnostic indicator of renalinjury or renal disease; an indicator of progression, remission, orrelapse of renal injury or renal disease; and an indicator of responseto treatment for renal injury or renal disease.
 3. The method of claim2, wherein periostin is detected as an indicator of epithelialmesenchymal transition (EMT).
 4. The method of claim 1, wherein theurine sample comprises a human urine sample.
 5. The method of claim 1,wherein the urine sample comprises centrifuged urine.
 6. The method ofclaim 1, wherein the urine sample comprises urinary exosomes.
 7. Themethod of claim 4, wherein the human is a human patient known to have,or suspected of having, renal injury or renal disease.
 8. The method ofclaim 7, wherein the human patient is known to have, or is suspected ofhaving, renal injury.
 9. The method of claim 7, wherein the humanpatient is known to have, or is suspected of having, acute renaldisease.
 10. The method of claim 7, wherein the human patient is knownto have, or suspected of having, chronic renal disease.
 11. A method ofdetecting an indicator of a subject's response to treatment for renalinjury or renal disease, the method comprising assaying a urine sampleobtained from a subject after initiation of treatment for renal injuryor renal disease for periostin, wherein the level of periostin ispositively correlated with the degree of renal injury or renal disease.12. The method of claim 11, wherein a baseline level of periostin ismeasured prior to initiation of treatment for renal injury or renaldisease.
 13. The method of claim 12, wherein the periostin level of theurine sample after initiation of treatment is compared to the baselinelevel of periostin.
 14. The method of claim 13, wherein a decrease inthe periostin level of the urine sample after initiation of treatment,as compared to the baseline level of periostin, indicates that thesubject is responding to the treatment.
 15. The method of claim 13,wherein one or more additional assays of periostin are performed astreatment is continued.
 16. The method of claim 1, additionallycomprising detecting one or more additional indicators of renal injuryor disease selected from the group consisting of serum creatinine, serumcystatin-C, urine protein, urine albumin, urineN-acetyl-beta-D-glucosaminidase, urine NGAL, IL-18, urine KIM1, andhematopoietic growth factor inducible neurokinin-1 (HGFIN).
 17. Themethod of claim 1, wherein periostin is detected by a method selectedfrom the group consisting of an immunoassay, HPLC, and massspectroscopy.
 18. The method of claim 11, additionally comprisingprescribing, initiating, and/or altering prophylaxis and/or therapy. 19.The method of claim 1, additionally comprising ordering and/orperforming one or more additional assays.
 20. The method of claim 19,wherein the additional assay comprises a different assay.